I. Field of the Invention
The disclosure relates to conversion of biomass to biofuel or other useful products. More particularly, the disclosure pertains to the generation of microorganisms having higher ethanol yields.
II. Description of the Related Art
Thermophilic bacteria have been engineered to produce ethanol from the cellulose and/or hemicellulose fractions of biomass. Examples of such thermophilic bacteria include Clostridium thermocellum and Thermoanaerobacterium saccharolyticum, among others.
Thermoanaerobacterium saccharolyticum is a thermophilic, anaerobic bacterium able to ferment hemicellulose but not cellulose. Wild-type strains produce ethanol, acetic acid and under certain conditions, lactic acid as the main fermentation products.
Thermoanaerobacterium saccharolyticum may consume a variety of sugars derived from the hemicellulose fraction of lignocellulosic biomass and convert them to acetate, lactate and ethanol. A number of strategies have been pursued to engineer this organism for increased ethanol production. Generally these strategies involve deleting genes for lactate production, acetate production or both. These strategies have helped improve the ethanol yield, but each approach has its own limitations.
The primary hydrogenase in T. saccharolyticum is called hfs, which has four subunits, hfsA, hfsB, hfsC and hfsD. Complete deletion of all four subunits (A, B, C and D) has been reported (Shaw et al. 2009). Deletion of hfs (A, B, C and D subunits) does not increase ethanol yield. Deletion of hfs and ldh in combination increases ethanol yield by 44%. In a different approach, a mutant hfs operon (called hfs*), containing several point mutations from a high-ethanol-producing strain, is re-introduced into the wild-type strain. The resultant strain shows 36% increase in ethanol yield as compared to the parental strain (Shaw et al. 2015).